In vivo Macroscopic Fluorescence Lifetime Molecular Optical Imaging ABSTRACT There is still great need in better characterizing new targeted therapies in vivo, especially prior to clinical translation. In this regard, preclinical molecular imaging is a central tool in the targeted drug development pipeline. However, there is still a lack of integrated imaging platforms that can enable longitudinal (multiple time points) and spatially-resolved monitoring of complex fingerprints including molecular, metabolic and functional signatures in the same tumor/subject. This new integrated multiplexing imaging platform will play a crucial role in the development of the next generation of targeted drugs and elucidating (multi-) drug resistance mechanisms. Recently, we have demonstrated the unique capabilities of optical imaging in quantifying receptor-target engagement in live subjects by leveraging fluorescence lifetime. This outstanding achievement was realized thanks to the combination of instrumental, algorithmic and biochemical innovations to enable, for the first time, whole-body time-resolved optical imaging based on structured light for 2D or 3D Frster resonance energy transfer (FRET) imaging in live subjects. Herein, we will further impact the field of optical preclinical imaging and drug delivery assessment by 1) integrating a cutting-edge high-resolution, time-resolved SPAD array imager for improved photon collection efficiency, spatial resolution, and portability; 2) we will harness the latest developments in Deep Learning (DL) for ultra-fast, quantitative, but fitting/iterative inverse solver-free image formation, in both 2D and 3D providing image formation/processing solutions to facilitate multiplexed imaging; 3) we will implement new functionalities in our imaging platform to enable the concurrent longitudinal imaging of multiple clinically relevant target-receptor interactions (e.g. HER receptor family members involved in many cancers) as well as metabolic and functional status across the whole-tumor region in live intact animals.